I’ve been given a scenario in which I must determine the Linking (L), ΔLinking (ΔL), Twisting (T), and Writhing (W) values for a plasmid after several modifications and I’m having a bit of trouble. The class does not have a book associated with it, and the searches I’ve done have yielded either basic definitions or advanced articles.
The plasmid is 5000bp and initially completely B-DNA (10bp/turn) and has 25 underwindings.
I set up a reference state of L = +500, ΔL = 0, T = +500, W = 0. Assuming I’m doing this correctly, the inital state is then L = +475, ΔL = -25, T = +500, and W = -25.
The first step involves nicking the plasmid once in once strand. This relaxes the DNA, so I think my values are then identical to the reference state for Step 1.
Step 2 involves changing a 240bp region to Z-DNA by changing the salt concentration for this region(I am given that Z-DNA has 12bp/turn, is left-handed, sugars alternate between 2′- and 3′-endo, and the bases alternate between syn and anti conformation). My values for this step (for the entire plasmid) are then L = +456, ΔL = -44, T = +456, W = 0.
In step 3, the plasmid is re-ligated (the nick is re-sealed). I am not sure if in this case W will still be 0, or if it will return to -25…
Finally, in step 4, the salt concentration is returned to its original state, so the entire plasmid is once again B-DNA. I think W would be unaffected by this, and I’d only have to calculate the new value for T (and this would change ΔL).
If anyone can offer me help (especially with Step 3) or can confirm that I am approaching this correctly I would greatly appreciate it.